Non-Coding RNAs

ncRNAs are RNA molecules that do not encode proteins but exert regulatory, structural, and catalytic roles within the cell.

• They influence transcription, RNA processing, translation, genome defence, and chromatin organisation.

  • Small ncRNAs: <200 nucleotides.

  • Long ncRNAs (lncRNAs): >200 nucleotides (often several kilobases).

Small ncRNAs typically range from 20–30 nucleotides and act with protein partners—especially the Argonaute family—to mediate gene silencing.

Major Classes of Small ncRNAs:

siRNAs (Small Interfering RNAs):

~21 nt, derived from long double-stranded RNA (dsRNA).

• Sources:

  • Exogenous (e.g., viral dsRNA).

  • Endogenous (e.g., convergent transcription, repetitive elements).

• Perfect complementarity to target mRNA.

  • Central to the RNA interference (RNAi) pathway.

    • Function:

      • Target cleavage via Argonaute-2 (AGO2).

      • Antiviral defence.

      • Generation of heterochromatin through amplification by RNA-dependent RNA polymerase (RdRP).

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miRNAs (MicroRNAs):

Typically ~22 nt, and endogenously encoded within the genome.

• Regulate development, differentiation, and homeostasis.

  • Origin:

    • Processed from pri-miRNA hairpins transcribed by RNA polymerase II.

  • Function:

    • Bind mRNAs with imperfect complementarity (especially in animals).

      • Cause:

        • Translational repression.

        • mRNA destabilisation.

        • Endonucleolytic cleavage (mainly in plants).

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piRNAs (PIWI-interacting RNAs):

24–32 nt, longer than siRNAs/miRNAs.

• Partner with PIWI proteins (subset of Argonautes).

  • Specific to germline cells.

    • Function:

      • Suppress transposable elements.

      • Maintain genomic integrity during gametogenesis.

  • Distinctive mechanism:

    • Dicer-independent biogenesis.

    • Ping-pong amplification cycle.

Core Protein Machinery:

Small RNA pathways rely on conserved protein complexes.

Dicer (RNase III Endonuclease):

• Converts long dsRNA or precursor miRNA hairpins into small RNAs.

  • Key structural domains:

    • dsRNA-binding domain (dsRBD) – recognises dsRNA substrates.

      • RNase III domains (RNase IIIa/IIIb) – catalyse cleavage to produce duplex siRNA/miRNA.

        • PAZ domain – binds 3’ overhangs of small RNA duplexes.

          • ‘Molecular ruler’ region – determines product length (e.g., ~21–23 nt in animals).

  • Organismal differences:

    • Mammals: Dicer-1 (miRNAs), Dicer-2 (siRNAs).

    • Invertebrates: Typically a single multifunctional Dicer.

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Argonaute Proteins (AGO Family):

• Core component of the RISC (RNA-Induced Silencing Complex).

  • Domain architecture:

    • PAZ – binds the 3′ end of guide RNA.

    • MID – anchors the 5′ phosphate of the guide.

    • PIWI – RNase-H-like endonuclease responsible for mRNA cleavage (“slicer” activity).

  • Functions:

    • AGO2: Only human Argonaute with robust slicer activity.

    • Guide selection: One strand (passenger) discarded; the guide strand directs targeting.

Mechanisms of RNA Interference:

siRNA Pathway:

• dsRNA → Dicer → siRNA duplex → AGO2 loading → target cleavage.

  • Features:

    • Perfect base pairing with targets.

    • Can initiate RdRP-mediated amplification (in plants, nematodes).

    • Important for antiviral defence and heterochromatin formation.

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miRNA Pathway:

• Biogenesis

  • pri-miRNA transcribed by RNA Pol II.

    • Processed by Drosha–DGCR8 (Microprocessor) in the nucleus, generating pre-miRNA.

      • Exported via Exportin-5.

        • Cleaved by Dicer, forming an miRNA duplex.

          • Loaded into AGO forming miRISC.

• Modes of Action

  • Translational repression (initiation block or slowed elongation).

    • mRNA deadenylation and decay.

      • Endonucleolytic cleavage (mostly in plants where complementarity is higher).

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piRNA Pathway:

• Dicer-independent; produced from long single-stranded precursors.

  • Amplified via ping-pong cycle between Aubergine (AUB) and AGO3.

    • Ensures:

      • Transposon silencing.

      • Epigenetic control of germline genome.

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Short Hairpin RNAs (shRNAs):

• Engineered analogue of endogenous RNAi molecules.

  • Expressed from plasmids or viral vectors.

    • Fold into hairpins processed by Drosha → Dicer.

      • Widely used in:

        • Gene silencing experiments.

        • Cancer gene therapy.

        • Antiviral strategies.

        • Agricultural virus resistance.

Long Non-Coding RNAs (lncRNAs):

LncRNAs exceed 200 nt and show highly diverse structures and functions.

Structural Complexity:

• Primary structure: Linear sequence with regulatory motifs (e.g., G-quadruplexes).

• Secondary structure: Stem-loops resembling tRNA-like or mRNA-like structures.

• Tertiary structure: Complex folding with protein partners (e.g., PRC2) or DNA interaction.

  • Capable of forming:

    • Ribonucleoprotein complexes (RNPs).

    • DNA–RNA hybrids.

    • Chromatin-associated scaffolds.

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Key Examples:

Xist (X-Inactive Specific Transcript):

• Located on the X chromosome.

  • Essential for X-chromosome inactivation (XCI) in placental mammals.

    • Functions:

      • Coats the X chromosome in cis.

      • Recruits chromatin-modifying complexes (e.g., PRC2).

      • Anchors inactive X to the nuclear lamina.

      • Establishes heterochromatin → transcriptional silencing.
        

H19 lncRNA and IGF2 Imprinting:

• Classical example of genomic imprinting.

  • H19 expressed from the maternal allele; IGF2 from the paternal allele.

    • Mechanism:

      • H19 lncRNA competes for enhancer interactions.

      • DNA methylation of Imprinting Control Region (ICR) dictates allele-specific expression.
        

HOTAIR (HOX Transcript Antisense RNA):

• Transcribed from HOXC locus, acts in trans on HOXD locus.

  • Recruits:

    • PRC2 → H3K27 trimethylation (gene silencing).

    • LSD1/CoREST → H3K4 demethylation.

Major regulator of developmental gene expression and oncogenic pathways.

Reverse Transcription and cDNA:

Reverse transcription converts RNA → DNA, opposing the central dogma.

• Performed by RNA viruses, especially retroviruses (e.g., HIV).

  • Reverse transcriptase (RT):

    • Synthesises complementary DNA (cDNA) from RNA.

    • Contains RNA-dependent DNA polymerase and RNase H activities.

    • High error rate → promotes viral evolution.

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Mechanism of Retroviral DNA Synthesis:

• Viral RNA contains long terminal repeats (LTRs): U3, R, U5.

  • A host tRNA primes DNA synthesis at the PBS (primer binding site).

    • Steps:

      1. Minus-strand DNA synthesis initiated by tRNA primer.

      2. RNase H degrades RNA template except for specific fragments.

      3. Strand transfer events allow completion of full-length DNA.

      4. Plus-strand synthesis occurs using remaining RNA fragments as primers.

      5. Final product: integrase-ready double-stranded viral DNA.

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Laboratory Reverse Transcription:

• Used to convert RNA → cDNA for:

  • RT-PCR.

  • Transcriptomics.

  • Gene expression quantification.

• Requires:

  • Reverse transcriptase enzyme.

  • dNTPs.

  • RNA template.

  • Primers: random hexamers, oligo-dT, or gene-specific.

• Modern engineered RTs:

  • Increased thermostability (~50 °C).

  • Lower error rates.

  • Improved sensitivity for low-abundance transcripts.

Application of RNA Technologies:

• RNAi-based pesticides targeting insect or viral genes (high specificity).

• Therapeutic shRNAs in gene therapy.

• piRNA pathway engineering for genome defence.

• lncRNA targeting in cancer epigenetic therapies.

• mRNA vaccines:

  • Rapid redesign to match viral mutations.

  • Avoids need for pathogen cultivation.

  • Encodes stable antigens despite rapid viral evolution.